This application addresses broad Challenge Area (06) Enabling Technologies, and specific Challenge Topic, 06-NS-106: Validating new methods to study brain connectivity. Mapping the structure and function of neural circuits is an important prerequisite to understand how groups of interconnected neurons produce perceptions and drive behavior. One challenge in mapping neural circuits is to unambiguously identify synaptic partners. Traditionally, synaptic connectivity has been studied using electrophysiology and electron microscopy - methods that provide critical detail but are impossible to apply in large scale. We aim to develop and validate a system to more easily identify synapses between selectively tagged neurons in the mouse. Recently, a system named GFP Reconstitution Across Synaptic Partners (GRASP), has been developed in invertebrates to study synaptic connectivity. It relies on genetic expression of two non-functional, complementary GFP fragments that are exposed on the extracellular sides of different cell populations. GFP reconstitution, and therefore fluorescence, occurs at the sites of close contact (e.g. synapses) between these cells. GRASP has many advantages: it can be genetically targeted to specific neuronal populations, it is a fluorescent system that can be readily visualized using traditional microscopy, and can be easily adapted to answer a wide variety of different questions about synaptic connectivity of neurons. To validate this technology for use in mammalian systems, we will initially test a battery of GRASP constructs in cell culture and an insect model. The most promising combinations will then be used to generate general-use transgenic lines that can be employed in concert with the Cre/LoxP and the tet-TTA systems to control expression of GRASP in time and space. In addition, we will develop viral carriers for GRASP as an alternate means of delivery and spatial restriction. We will use GRASP to help address questions of connectivity in the mammalian taste system, thereby providing validation of its utility to study mammalian neural circuits. Ultimately we anticipate that the genetically engineered GRASP mouse lines and viral vectors generated in this study will provide a toolbox that will be of considerable value for the entire neuroscience community. PUBLIC HEALTH RELEVANCE: Mapping the structure and function of neural circuits is an important prerequisite to understand how groups of interconnected neurons produce perceptions and drive behavior. We aim to develop and validate a system to more easily identify synapses between selectively labeled neurons in the mouse. We anticipate that our genetically engineered GRASP mouse lines and viral vectors will provide a toolbox that will be of considerable value for the entire neuroscience community.